RNA Pull-Down Protocol of CircRNA Research

Experiment Summary

The function of biomolecules is often realized through interactions with other molecules. CircRNAs, as widely present and highly diverse molecules in the cell, the study of their interactions with other molecules, including the dynamic regulatory process, is an important breakthrough in understanding their functions.

Currently, the study of circRNAs mainly focuses on their interactions with miRNAs or proteins, and the relevant experimental techniques include RNA fluorescence in situ hybridization, luciferase reporter gene assay, RIP technology, RNA pull-down technology, etc. Among them, RNA pull-down technology is the most effective. Among them, RNA pull-down technology, which can not only verify the molecules that may interact with each other, but also realize the exploration of unknown interacting molecules, has unique advantages in circRNA function research.

Materials and Reagents

Sterile Phosphate Buffer Saline (PBS).
Cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40).
Buffer A (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 % NP-40).
Streptavidin agarose beads or streptavidin magnetic beads.
tRNA (0.1 μg/μl).

Procedure

A. Preparation of cell lysates

Harvest cells by centrifugation at 1000g for 5min at 4°C using one plate of cells (~10⁷) and discard the supernatant.
Resuspend the precipitate by adding 1ml of ice-cold PBS and centrifuge at 1000g for 5min at 4°C.
Resuspend the precipitate by adding 500 Cell lysis buffer (+ fresh protease inhibitor, RNAse inhibitor, DTT) and place on ice for 5min, mixing several times during the period of inversion.
Centrifuge at 13000g for 10min at 4°C.
Collect the supernatant for direct use in experiments or add 10% glycerol and store at -80°C, discard the cell debris.

QC: Detect the presence of target proteins in cell lysate as a Western assay. RT-PCR detects target RNA fragments in cell lysate.

B. Preparation of biotin-labeled RNA

Biotin-labeled DNA probes can be synthesized directly at the 5' or 3' end, and the biotin-labeled DNA probes can bind to specific circRNAs.

RNA Pull-Down Protocol of CircRNA Research

C. RNA pull-down

Take 150 μl streptavidin agarose beads and wash the beads with Buffer A to remove the stock solution.
Prepare precleared cell lysate: Incubate cell lysates with 60 μl streptavidin agarose beads at 4°C for 30 minutes rotationally.
Centrifuge at 2500 rpm for 30s, retain the supernatant and add 1x volume of buffer A (+tRNA 0.1μg/μl). Then add 3 μg biotin-labeled RNA to precleared cell lysate. Incubate in rotary shaker at 4°C for 1 h by turning over.
Purification of biotin-labeled RNA-bound protein: add 30 μl of washed streptavidin agarose beads, rotate and incubate for 45 min at 4°C.
Wash the beads five times using buffer A.
Proteins were eluted for downstream analysis.

D. Downstream analysis

MS: PBS (+100μl 2mM biotin) was incubated with beads at room temperature for 30min with gentle shaking every few minutes, to obtain eluted proteins. The eluted proteins can be precipitated using TCA and digested by trypsin. Then mass spectrometry was performed.
MS detection of specific bands: Protein loading buffer was added to beads, separated on Bis-Tris gel with a gradient of 4-12%, silver stained using mass spectrometry compatible silver staining kit, and the corresponding strips were cut for mass spectrometry detection. The corresponding gel strips were cut and detected by mass spectrometry.
Western: Add protein loading buffer to beads, boil and denature for 5 min, separate on Bis-Tris gel with a gradient of 4-12%, and then do Western to detect the corresponding proteins.

* For research use only. Not intended for any clinical use.

Quick Inquiry

RNA Pull-Down Protocol of CircRNA Research

Experiment Summary

Materials and Reagents

Procedure

Related Products and Services at Creative Biogene